The objective of this proposal is to determine and compare the cellular pharmacology and the molecular actions of the three new cytosine nucleoside analogs 2'-C-Cyano-1-beta-D-arabinofuranosylcytosine (CNDAC), 2'-fluoromethylene-2'-deoxycytidine (FMDC) and 3'-ethynylcytidine (ECyd) with a view to understanding the mechanisms for their cytotoxic activity. The experimental therapeutics of these nucleosides in tumor-bearing animals and their toxicologic characteristics have been extensively investigated to bring then to clinical evaluation, which is ongoing. We hypothesize that a detailed knowledge of their cellular metabolism, mechanisms of action, and the application of this understanding to the design of strategies for combinations will enhance the effective use of these compounds. l) We will investigate the hypothesis that once the CNDAC nucleotide has been incorporated into DNA and a subsequent nucleotide has been added, an electronic rearrangement characterized as beta-elimination will occur, causing a break in one DNA strand. During this rearrangement, the analog in the 3'terminus would be converted to a de facto chain terminator. We will characterize the ability of 3' to 5' proof-reading exonucleases associated with DNA polymerases delta and epsilon, to remove the analog from the 3'terminus. CNDAC-treated cells arrest cell cycle transit in G2. We will investigate the molecular basis for invoking the G2 checkpoint and will evaluate strategies to kill cells by abrogating the checkpoint. Finally, CNDAC nucleotide is an excellent substrate for incorporation into DNA. We will compare its pharmacodynamics with those of other nucleotide analogs in quiescent cells induced to undergo DNA repair. 2) Our data demonstrate that the major activity of FMDC is due to incorporation into DNA. We will characterize the kinetics of FMDC nucleotide incorporation in model systems, and we will evaluate the ability of proof reading exonucleases to excise it, and will characterize its ability to serve as a substrate for DNA ligase. Because FMDC is efficiently incorporated into DNA, we will evaluate its interactions with DNA damaging drugs in quiescent cells. 3) Preliminary studies demonstrate that ECyd acts as a ribonucleotide analog, with it majors actions on RNA transcription. We will characterize the cellular metabolism of ECyd. Strategies to enhance its incorporation into RNA, thereby potentially amplifying its actions, will be tested. The specificity of ECyd nucleotides against specific RNA polymerases will be studied. Cells that require a specific protein factor for survival may be selectively affected by inhibition of the transcription that sustains the actor. A model will be designed and tested with ECyd, and compared with other transcription and translation inhibitors.